Techniques for freezing spermatogonia cells

ABSTRACT

Methods for cryopreserving spermatogonia cells are presented.

The research on which the technology described in this document is basedwas supported by U.S. government funding. The present invention is basedon work in part funded by the National Institutes of Health and theUnited States Department of Agriculture.

This application is a Continuation-in-Part of application Ser. No.08/345,738, filed Nov. 21, 1994, which is a Continuation-in-Part ofapplication Ser. No. 07/987,250, filed Dec. 7, 1992, now abandoned whichis a Continuation-in-Part of application Ser. No. 07/802,818, filed Dec.6, 1991, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to animals harboring a non-native germcell, to corresponding animal lines and germ cells, and to methods forobtaining the same. The present invention provides methods forcryopreserving spermatogonia cells.

2. Discussion of the Background

There have been many attempts to influence differentiation of developingcells by modifying the genotype of an embryo and then observing itseffect on the phenotypic development pattern in the progeny. Thesetechniques included transgenic methods (Brinster et al, Harvey Lectures,80:1-38 (1986) nuclear transfer (McGrath & Solter, Science (1983)220:1300-1302) and cell-egg fusions (Graham, "Heterospecific GenomeInteraction," 1969 Wistar Institute Press, pp. 19-35, ed. Defendi). Ofthese, the latter two have had limited success.

Another approach might be to add a stem cell(s) to an early embryo anddetermine its effect on development. For example, one might imagine thata stem cell from bone marrow would contribute to the population of, andthus modify the differentiation of the evolving bone marrow cells in thehost embryo. To pilot these experiments, older embryo cells wereintroduced into young embryos resulting in modest success withcolonization (Moustafa & Brinster, J. Exp. Zool. (1972) 181:193).

These initial experiments were followed by studies using bone marrowstem cells and teratocarcinoma stem cells (also called embryonalcarcinoma cells). There was evidence that both these cells colonized thedeveloping embryo (Brinster, J. Exp. Med. (1974) 140:1049-1056). In thecase of the embryonal carcinoma (EC) cell, the colonization wasdemonstrated dramatically by a change in the color of hairs in the coatof the mouse. This was an exciting result which stimulated a great dealof interest among scientists in the field, because it showed thepossibility of colonizing an animal with non-embryo cells. This wouldprovide a means to introduce new genetic information through the DNA ofthe colonizing cells.

The next year these results were confirmed and extended by two otherlaboratories (for work done in one of these laboratories, see Mintz &Illmensee, Proc. Nat. Acad. Sci. (USA) (1975) 72:3585-3589) and it wasdemonstrated that the introduced EC cells may colonize numerous tissuesincluding germ cells (sperm and eggs). Thus, a gene that was mutated,modified, or added to the cell in vitro could eventually end up in spermor eggs of an animal, creating a new genetic strain of mice.

Unfortunately EC cells colonized the germline poorly and a better cellline was sought. In 1981 two scientists, Gail Martin and Martin Evans,independently described a more efficient cell designated the embryonicstem (ES) cell (Martin, Proc. Nat. Acad. Sci. (USA) (1981) 78:7634;Evans & Kaufman, Nature (1981) 292:154). These cells colonize thegermline better than EC cells. However, it seems likely that they arisefrom the same pool of primitive cells in the embryo and are quitesimilar in biological characteristics.

Embryonic stem cells can be modified in vitro (in culture flasks) byadding genes or changing endogenous genes and then the modified cellsintroduced into a blastocyst where they participate in development andcan become sperm. This technique allows very specific modification ofthe mouse genome and perhaps other species.

Techniques for obtaining non-human transgenic animals through theinjection of DNA into eggs are also known. See e.g., Gordon et al, Proc.Nat. Acad. Sci. (USA), (1980) 77:7380-7384. These techniques however, aswell as the use of ES cells noted above, are very labor intensive.

Gene therapy is by definition the insertion of genes for the purpose ofmedicinal therapy. The principle underlying gene therapy is to, ratherthan deliver doses of one or more pharmacologic molecule, deliver afunctional gene whose RNA or protein product will produce the desiredbiochemical effect in the target cell or tissue. The genes may bedelivered into endogenous cells or within new cells delivered to theanimal. There are several potential advantages of gene therapy overclassical biochemical pharmacology, including the fact that insertedgenes can produce extremely complex molecules, including RNA andproteins, which can be extraordinarily difficult or impossible toadminister and deliver themselves.

The many applications of gene therapy, particularly via stem cellgenetic insertion, have been extensively reviewed (see, Boggs et al.,Int. J. Cell Cloning (1990) 8, 80; Kohn et al., Cancer Invest. (1989) 7,179; Lehn, Bone Marrow Transpl. (1990) 5. 287; and Verma et al.,Scientific Amer. (November 1990) 68). Genetically transformed human stemcells have wide potential application in clinical medicine, as agents ofgene therapy.

Methods and compositions are known for the ex vivo replication andstable genetic transformation of animal, including human, stem cells andfor the optimization of such stem cell cultures. The applications ofgene therapy could be advantageously expanded if techniques wereavailable for the application of gene therapy to animal primitive germcells.

SUMMARY OF THE INVENTION

Accordingly, it is an object of this invention to provide a facilemethod for obtaining animals harboring a biologically functionalnon-native, preferably male, germ or stem cell, and for obtainingcorresponding resultant germ or stem cells.

It is another object of this invention to provide a facile method forobtaining new animal lines, and for obtaining corresponding resultantgerm or stem cells.

It is another object of this invention to provide animals harboring abiologically functional non-native germ or stem cell, their progeny, andcorresponding resultant animal lines and germ or stem cells.

It is another object of this invention to use as said non-native germcell, one or more animal stem cells which has been cultured maintainedand/or expanded in culture ex vivo.

It is another object of this invention to provide methods forcryopreserving spermatogonia cells.

It has been discovered by the inventor that the above objects and otherobjects which become apparent from the description of the inventiongiven hereinbelow are satisfied by the following method. Primitive germcells are obtained and are optionally expanded and/or modified ex vivo.The testis (or testes) of a male animal host is (are) repopulated withat least one primitive cell (e.g. a totipotent stem cell) which is notnative to the host. Because the donor cell(s) is (are) stem cells, theycan expand to repopulate the seminiferous tubules of the recipienttestis. The recipient animal may then be bred to obtain a novel animalline in which every cell of the descendant animal is geneticallynon-native to the original recipient host animal. The methods of thepresent invention have now been successfully used to produce progenythat contain genetic information different from the biological sire(father). That is, the genetic information in the progeny is that of thedonor stem cell and not that of the recipient male which is the natural(biological) sire.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 and 2 are photographs of cross-sections of seminiferous tubulesfrom the testes of adult mice. FIG. 1 is a control. FIG. 2 comes from atransgenic (ZFlacZ) mouse in which late stages of spermatogenesis stainblue and hence can be used as a marker for stem cell differentiation tomature spermatozoa. FIG. 2 shows intense blue (dark) staining in allmature stages of spermatogenesis, which occupy the center of the tubule.Control FIG. 1 does not. Stem cells taken from an animal carrying theZFlacZ transgene (FIG. 2) will stain blue if they are transferred to arecipient, survive and differentiate into spermatozoa. Staining does notoccur in the cells of the recipient male, because it does not carry themarker transgene ZFlacZ.

FIG. 3A-D are photographs of a cross-section of testes, showing blue(dark) stained tubules, demonstrating, survival, differentiation anddevelopment of transferred cells that carried the ZFlacZ transgene. FIG.3B is a higher magnification view of the tubules shown in FIG. 3A. FIGS.3C and D likewise show blue (dark) stained tubules.

FIGS. 4A and B are photographs demonstrating spermatogenesis in thetestis of a recipient male following transfer of ZFlacZ donor cells. A)Testis from (C57Bl/6 x SJL) F1 Busulfan®-treated male 6 months afterdonor cell transfer. Note active spermatogenesis and mature spermatozoa.Round spermatids and more mature stages stain blue. When stain isintense, immature stages also appear blue. B) Tubules from epididymis ofmouse with testis shown in A). Many mature spermatozoa appear as reddots in lumen of the tubules. Background blue color in the lumen is theresult of β-galactosidase (transgene) activity arising from thecytoplasm of immature sperm cells. Mature spermatozoa do not stain forβ-galactosidase because they lack sufficient cytoplasm. Stain: X-galfollowed by neutral fast red. (A x 280; B x 125)

FIGS. 5A and B are photographs of testis of Busulfan®-treated recipientmale injected with ZFlacZ donor cells 14 months previously. A) Grossappearance. The testis is approximately 80% normal size, because aninadequate Busulfan® effect permitted the reinitiation ofspermatogenesis from endogenous stem cells (unstained tubules). Bluetubules indicate spermatogenesis from ZFlacZ donor spermatogonial stemcells. Testes has been partially transacted longitudinally to allow forpenetration of fixative and stain (X-gal). Scale bar 1 mm. B)Microscopic appearance. Blue tubule cross sections representspermatogenesis from donor stem cells. Red tubule cross sections reflectspermatogenesis from endogenous stem cells. The ratio of blue to redtubules across several complete testes cross sections in this area wasapproximately 1:12. Other areas of the testis have fewer blue tubules.Stain: X-gal followed by neutral fast red (x 70);

FIG. 6 are testes from progeny of the recipient male described in FIG.5. The testis on the left stained blue with X-gal indicating thepresence of the transgene in all cells. The testis on the right is fromanother offspring and is not blue because it does not contain thetransgene. The seminiferous tubules of the parent male displayed amosaic pattern of staining resulting from donor stem cell and endogenousstem cell areas of spermatogenesis (See FIG. 5). Age of progenyapproximately 10 weeks. Born 11 months following donor cell transfer.Scale bar 0.25 cm.

FIG. 7 is a diagram of the procedure used in example B.

FIG. 8 is a photograph showing the morphology of testes withtransplanted stem cells previously stored at -196° C. FIG. 8A. Testis ofdonor mouse (designated ZFlacZ) which carries an E. coli β-galactosidasetransgene (lacz) that allows round spermatids and later stages ofspermatogenesis to be stained blue following incubation with5-bromo-4-chloro-3-indole-β-D-galactosidase (X-gal). FIG. 8B. Testis ofrecipient male (C57BL/6 x SJL) F1 treated with busulfan (36 mg/kg) todestroy endogenous spermatogenesis. FIG. 8C. Left testis of recipientmale 891 (Table 2) that received donor testis cells isolated from adultmice (8-16 wks of age) and frozen 7 days. The testis has been bisectedto allow penetration of fixative and stain. FIG. 8D. Left testis ofrecipient male 771 (Table 2) that received donor testis cells isolatedfrom prepubertal mice (6-14 days of age) and frozen 111 days. Followingincubation with X-gal, blue tubules in C and D identify areas ofspermatogenesis from frozen donor cells. Scale bar, 1 mm.

FIG. 9 is a photograph showing the microscopic appearance ofspermatogenesis in recipient seminiferous tubules followingmicroinjection of donor testis cells preserved at -196° C. FIG. 9A.Seminiferous tubule of donor testis from transgenic ZFlacZ mouse. Roundspermatids and more mature stages stain blue following incubation withX-gal (FIG. 1A). When staining is intense, immature stages also appearblue. FIG. 9B. Seminiferous tubule from recipient mouse treated withbusulfan. No germ cell stages are present. Only Sertoli cells remain.FIG. 9C. and 9D. Seminiferous tubules from busulfan treated recipientmice 891 and 771, respectively (Table 2). Blue staining of germ cellsindicates their origin from transplanted donor cells that had beenfrozen 7 and 111 days, respectively. Background stain in all sections isneutral fast red. Scale bar 50 microns.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

According to the present invention, primitive cells (e.g.,spermatogonial stem cells) are introduced into prepared seminiferoustubules of animal testis (or testes). There, the introduced primitivecells develop into mature spermatozoa. This colonization is possiblebecause the lumen of the seminiferous tubule is an immunologicallyprivileged site and provides the proper environment. Evidence for thisis the absence of endogenous antibody specific to sperm, and thepresence of T-cells that are tolerant to spermatozoa in the circulatingblood of males. Furthermore, injection of sperm into the tissues of amale generally provokes an immunological response.

Cells which can develop into more differentiated cells of two or moretypes are used as the primitive cells. Thus, one of two types of cellscan be used: (1) totipotent cells, which are cells having the potentialto differentiate into any cell type, including germ cells (e.g.,spermatogonial stem cells); and (2) pluripotent cells, which are cellscapable of differentiating into two or more types of cells, e.g., bonemarrow stem cells, liver stem cells, kidney stem cells, etc.

An illustrative procedure for repopulating the testis (or testes) of ahost is as follows. A male host animal is prepared by destroying thenative germ cell population in the seminiferous tubules by a method thatleaves intact (i.e., biologically functional) the supporting cells,including the Sertoli cells. Suitable known methods include physicalmeans (e.g. radiation¹, heat², etc.), chemical means (e.g. cadmium³,Busulfan®⁴, etc.), but any other known techniques to selectively destroynative sperm cells can be used. Preferably radiation is used, withBusulfan® being a useful chemical method.

The amount of native germ cell population destroyed by the abovetechniques may be varied depending on the outcome desired (e.g., partialreplacement or full replacement of endogenous spermatogenesis). Someapplications of the invention may be best served by partial replacement(see below). In a first embodiment, the total native germ cellpopulation is destroyed to ensure that every offspring sired by therecipient male will be genetically identical to the donor animal. In asecond embodiment, less than the total (e.g., between 10 and 90%) of thenative germ cell population of the recipient male is destroyed. In thissecond embodiment, the recipient male will produce both offspring whichare genetically identical to itself as well as offspring geneticallyidentical to the donor animal.

A typical Busulfan® treatment is illustrated as follows. Fortymilligrams of Busulfan® are dissolved in 5 ml of dimethylsulfoxide, towhich 5 ml of water are then added. An aliquot of this solution isinjected intra-peritoneally into a mouse in an amount sufficient toadminister a dose of 1 mg of Busulfan® per 25 g (40 mg/kg) of mouseweight (Bucci et al, Mutation Res. (1987) 176:259-268). The male is thenallowed to recover, and tested to ascertain that there is no remainingspermatogenesis by breeding and/or testicular biopsy.

Alternatively, in mice receiving lower dose of Busulfan® (e.g., eitherpurposeful by the researcher or by reason of leakage at the time ofinjection), tubules will repopulate with both the transferred germ orstem cell line as well as with surviving endogenous stem cells.

Alternatively, one can employ a male host that genetically has lownumbers or no sperm in its testes, such as inbred strains of mice thatproduce offspring with seminiferous tubules depleted of sperm cells. Forexample, the sperm cells of so-called W-mutant mice are deficient for anenzyme necessary for their survival and proliferation. Homozygotes andcompound heterozygmotes for certain alleles of this gene are sodeficient for this enzyme that they are completely devoid of sperm cellswhen adult. C57BL/6J mice segregating the W^(v) and W⁴⁴ alleles, WB/ReJmice segregating the W allele, and 129/SV mice segregating the W⁵⁴allele can be maintained and bred toward this end. For example, aC57BL/6J W^(V) /+ animal bred to a WB/ReJ W/+ will yield a proportion ofhybrid W/W^(v) males, which when mature will totally lack sperm cells.The testes of these hybrids may accept donor cells from C57BL/6J mice orWB/ReJ mice without histocompatibility problems.

If necessary, tolerance in the recipient can be induced by transferringcells corresponding to those primitive cells being transferred to thetestis (or testes) of the host, to the thymus of the host beforetransfer of the primitive cells to the testis (or testes). Thus ifspermatogonia is the primitive cell being transferred to the testis (ortestes) of the host, spermatogonia may be first transferred to thethymus of the host to induce tolerance. See, Posselt et al, Science(1990) 249:1293-1295.

Alternatively, cells from an animal of the same strain as the recipienthost can be used, or an immunodeficient host can be used. For an exampleof the latter, inbred strains of mice (e.g. SCID mice or nude mice) bredto act as recipients of sperm cells can be used. As an example of theformer C57 mice segregating the c (albino) and C (color) allele can bemaintained. Homozygotes and compound heterozygotes for these alleles areeasily distinguished by coat color. A Busulfan®-treated homozygote forthe one allele can receive donor cells from a homozygote for the otherallele. If the recipient animal thereafter gives rise to offspringshowing the donor-type coat color marker, this is evidence that thedonor cells gave rise to functional sperm.

The primitive cells used in accordance with the invention can come fromother individuals (including both the same and other species) or invitro culture. The primitive cells may be harvest from a fetus in itsthird trimester, a newborn, a preadolescent, a fertile individual, anolder individual who is clinically infertile but is still producingprimitive cells, or a recently deceased individual provided theprimitive cells are isolated prior to cell death (the cells arepreferably isolated within 2 hours of death). Examples of primitivecells that can be used include totipotent stem cells, embryonalcarcinoma cells, embryonic stem cells, sperm cells from other males(e.g. juvenile males with high levels of primitive sperm cell types),primordial germ cells, other primitive cells, etc. Primitive sperm cellsfrom seminiferous tubules, embryonic stem cells grown in culture, orprimitive cells from body organs are prime candidates. The use of female(XX) cells is also within the scope of the present invention.

These primitive cells may be obtained in accordance with knownprocedures. For example, spermatogonia stem cells may be obtained asfollows. The testis of a neonatal animal is collected and the coveringremoved. The tubules are enzymatically-digested to produce a single-cellsuspension which contains approximately 15% stem cells. Besides suchspermatogonial stem cells found in neonatal and adult testis, there areseveral other known sources of suitable cells. Stem cells from a tissueculture, such as embryonic stem and embryonal carcinoma(teratocarcinoma) cells are easily utilized. Other desirable cells areprimitive cells from organs which have the potential to regenerate, suchas the liver, or lymphoid organs, such as the bone marrow.

Alternatively, primitive cells may be obtained by a testicular biopsy inorder to preserve the fertility of the donor animal.

When the primitive cells are cultured prior to implantation, theprimitive cells may be maintained and/or expanded in a culture mediausing known techniques. A suitable culture medium would be Dulbecco'smodified Eagle's Medium (DMEM) plus 15% fetal calf serum. Other culturemedium containing higher levels of pyruvate and lactate (See Brinster,In: Growth, Nutrition and Metabolism of Cells in Culture, Vol. II, Ed.Rothblat & Cristofalo, (1972), 251-286, Academic Press). Primitive malegerm cells should be cultured at 32° C. because of their sensitivity tohigh temperature. Other cells could be cultured at higher temperaturedepending on individual characteristics (e.g., chicken cells at 37°-42°C.). Various growth factors, such as stem cell factor, basicfibroblastic growth factor, leukemia inhibitory factor, and others couldbe used to enhance cell proliferation. A gas composition of 0-10% CO₂ in90-80% air could be used. However, variations in concentrations of O₂might be applicable for some stages of culture. The period of culturecould be short (<1 hr) or indefinite depending on the applicationenvisioned. In addition, the cells could be frozen at -196° C. (liquidnitrogen) with cell cryopreservation techniques for a period of timebefore, during or after culture and prior to reintroduction into arecipient testis.

Cell densities which may be used are of from 10⁴ to 10⁹ cell per ml ofculture medium. The medium used to culture the primitive cells maycomprise three basic components. The first component is a general mediacomponent, the second is a serum component and the third is a specialfactor component. Standard known media components such as, for example,IMDM, MEM, DMEM, RPMI 1640, Alpha Medium of McCoy's Medium can be usedand can contain combinations of serum albumin, cholesterol and/orlecithin, selenium and inorganic salts.

The serum component may be present in the culture in an amount of atleast 1% (v/v), based on the volume of the medium. The serumconcentration is preferably about 15% (v/v). Standard serum can be usedsuch as horse serum, human serum, fetal calf serum, newborn calf serum,etc. Alternatively, the serum component can be replace by any of severalstandard serum replacement mixtures which typically include insulin,albumin, and lecithin or cholesterol (See, Iscove et al., Exp. Cell Res.(1980) 126, 121 and Dainiak et al., J. Clin. Invest. (1985) 76 1237).

Glucose concentration is usually maintained in the range of about 5 to20 mM. Lactate concentration is usually maintained 5 mM or above.Glutamine concentration is generally maintained in the range of fromabout 1 to 4 mM. Ammonium concentration is usually maintained belowabout 2.4 mM. The concentrations can be monitored by either periodic oron-line continuous measurements using known methods. See, e.g., Caldwellet al., J. Cell. Physiol. (1991) 147, 344.

Suitably, the cell cultures may be supplemented with various growthfactors as described, e.g., in U.S. Pat. No. 5,199,942 (Gillis et al.).Ideally, the cell cultures are supplemented with at least one cytokinesuch as an interleukin 1-6 (IL 1-6), GM-CSF, erythropoietin (EPO), mastcell growth factor (MCGF), basic fibroblast growth factor, transforminggrowth factor β (TGF-β), platelet derived growth factor (PDGF),epidermal growth factor (EGF), etc.

Culturing is typically carried out at a pH which is roughly physiologic,i.e. 7.0 to 7.6.

The nutrient media of the present invention can be exchanged three timesweekly, either as single exchanges or a one-half medium and serumexchange. Alternatively, the nutrient media of the present invention canbe replaced continuously or periodically.

Following culturing, the cells may be isolated by trypsin digestion andprepared for transfer by centrifugation and resuspension in concentratedform (10⁶ -10⁸ cells/ml).

In a preferred embodiment the cells and/or tubules are modified tofacilitate the colonization as well as diversity of cells that areeffective. For example, the primitive cells may be added with theircorresponding Sertoli cells to facilitate population, the surface of theprimitive cells may be treated with phytohemagglutinin (PHA) to make theprimitive cells more adherent (Mintz et al, Dev. Biol. 31:195-99), orthe primitive cells may be added with corresponding "carrier" spermcells to facilitate repopulation. Alternatively the tubules can besubjected to a limited enzymatic digestion to render them moreaccessible to the transferred primitive cells.

In an optional embodiment of the invention, the primitive cells aremodified genetically by any of a variety of known techniques⁵ so thatthe genetic characteristics of the resulting spermatozoa can bepredetermined. The primitive cells which are used in accordance withthis embodiment of the invention may also be native cells possessingnaturally induced mutations or variations or native cells possessingartificially induced mutations or variations.

The primitive cells of the present invention can be modified in vitro bya variety of techniques (e.g., see Gene Targeting: A Practical Approach,A. Joyner (1993), IRL Press. Genes which may be used with thisembodiment include genes containing a DNA sequence (or the correspondingRNA sequence may be used) encoding an intracellular, secreted, or cellsurface molecule which is exogenous to the animal donor. Exogenous, inthis context, means that the animal host either lacks a properlyfunctioning gene sequence or lacks the gene entirely.

Alternatively, vectors can be used to express the gene. Suitable vectorscontaining the DNA sequence (or the corresponding RNA sequence) whichmay be used in accordance with the invention may be an eukaryoticexpression vector containing the DNA or the RNA sequence of interest.Techniques for obtaining expression of exogenous DNA or RNA sequences ina host or cell are known. See, for example, Korman et al, Proc. Nat.Acad. Sci. U.S.A. (1987) 84, 2150.

This vector, as noted above, may be administered to the germ or stemcell being cultured in a retroviral or other viral vector (i.e., a viralvector) vehicle, a DNA or RNA/liposome complex, or by utilizingDNA-mediated gene transfer. Further, the vector, when present innon-viral form, may be administered as a DNA or RNA sequence-containingchemical formulation coupled to a carrier molecule which facilitatesdelivery to the host cell. Such carrier molecule would include anantibody specific to the cells to which the vector is being delivered ora molecule capable of interacting with a receptor associated with thetarget cells.

Cell-mediated gene transfer may be used in accordance with theinvention. In this mode, one relies upon the delivery of recombinantgenes into living organisms by transfer of the genetic material intocells derived from the host and modification in cell culture, followedby the introduction of genetically altered cells into the host. Anillustrative packaging cell line which may be used in accordance withthis embodiment is described in Danos et al, Proc. Natl. Acad. Sci.U.S.A. (1988) 85, 6460.

The retroviral vector vehicles used in accordance with the presentinvention comprise a viral particle derived from a naturally-occurringretrovirus which has been genetically altered to render it replicationdefective and to express a recombinant gene of interest in accordancewith the invention. Once the virus delivers its genetic material to acell, it does not generate additional infectious virus but doesintroduce exogenous recombinant genes to the cell.

In other viral vectors, the virus particle used is derived from othernaturally-occurring viruses which have been genetically altered torender them replication defective and to express recombinant genes. Suchviral vectors may be derived from adenovirus, papillomavirus,herpesvirus, parvovirus, etc.

The molecules which may be used in accordance with this inventioninclude genes encoding immune stimulants such as Class Ihistocompatibility genes, Class II histocompatibility genes, bacterialgenes including mycobacterial (PPD) genes, genes encoding heat shockproteins, viral glycoproteins encoding genes including vesicularstomatitis virus G protein, influenza hemagglutinin, and herpes virusglycoprotein β, minor histocompatibility antigens; genes encoding growthstimulants/inhibitors including inducers of differentiation such asstimulants, including interleukin-2 (IL-2) IL-4, 3, 6 or 8,inhibitors/inducers of differentiation, such as TNF-α or β, TGF-β (1, 2or 3), IL-1, soluble growth factor receptors (PDGF, FGF receptors),growth factors and analogs (PDGF, FGF), interferons (α, β or γ),adhesion molecules, etc.; gene encoding selectable markers includingthymidine kinase, diphtheria toxin, pertussis toxin or drug-sensitiveproteins.

The DNA/RNA sequence is preferably obtained from a source of the samespecies as the animal recipient, but this is not absolutely required,and the present invention provides for the use of DNA sequences obtainedfrom a source of a species different from the donor or recipient animalin accordance with this embodiment.

These genes are suitably operably linked to appropriate elementsincluding promoter, enhancers, repressors, etc. Techniques known in theart can be used to select suitable elements and to operably link them tothe above genes.

The selected primitive cells are then introduced into the individualtubules. For example, a prepared male can be anesthetized and the testis(or testes) surgically exposed. By micromanipulation methods a thinglass needle is introduced into exposed tubules, one after another, andeach tubule is injected with a solution containing the primitive cellsbeing used to colonize the tubule. Alternatively, the primitive cellscan also be introduced by injecting into other parts of the tubularsystem e.g. the lumen of the rete testes. To inject the rete testes onemay use either fine stainless-steel needles or fine pulled-glasscapillaries loaded with donor primitive cells. A micromanipulator isused to direct the tip of such an instrument to penetrate the retetestis or the seminiferous tubule. The cells are expelled and willback-fill the seminiferous tubules.

In a preferred embodiment, to minimize the number of injection sites andto increase the efficiency of injection of a concentrated primitive cellsuspension, a glass pipette with a 1 mm outside diameter and a 40μ tipis secured into a micropipette holder. The tip of a 1 ml plastic syringecan then be inserted into the other end of the micropipette holder, andthe syringe cut at about the midlength mark. The cell solutioncontaining the primitive cells is then deposited in the syringe barrel,which is then screwed onto the metal end of an Eppendorf capillaryholder attached by tubing to a pressure injector. To expose the tubules,only 1 to 3 small incisions (1-3 mm) need be made in the tunica pertestis. using this embodiment, an average of 80 percent of the surfacetubules can be filled with concentrated cell suspension.

The cell suspension of primitive cells suitably comprises an injectionmedium and primitive cells (e.g., 10⁶ -10⁸ cells per ml). For example,the injection media can comprise NaCl, Na₂ HPO₄, KCl, KH₂ PO₄, EDTA,pyruvate, lactate, glutamine, glucose, bovine serum albumin, and DNAseI. The pH of the injection media is suitably in the range of 7-7.7,preferably 7.4.

Other systems may also be suitably used for introduction of the cells.These include injection into the vas deferens and epididymis ormanipulations on fetal or juvenile testes, techniques to sever theseminiferous tubules inside the testicular covering, with minimumtrauma, which allow injected cells to enter the cut ends of the tubules.For example, a fine surgical thread is circled about a number oftubules, and then drawn tight, severing the tubules. A donor-cellsuspension is then injected into the testis.

Alternatively neonatal testis (or testes), which are still undergoingdevelopment, can be used. Here, a surgical procedure to expose theneonatal testis (or testes) for injection of new cells is used. Neonatalmice are chilled on ice for anesthetization. The tiny testes aresurgically exposed, and a small bladed-instrument is used to disrupt thetubular architecture. Donor cells are injected, and become incorporatedduring repair. These cells then participate, with endogenous cells, ifpresent, in the maturation of the testis.

As noted supra, the primitive cells entering the tubule are generallyprotected from destruction by the immunologically privileged environmentof the internal lumen of the tubule. Cells that leak from the tubule aretypically destroyed by the immune system of the host since the cells areforeign to the animal.

The destruction of cells outside the tubule lumen is desirable becauseprimitive stem cells have a high propensity to undergo transformationinto malignant growths. But if this undesirable property is eliminatedfrom the primitive cells then destruction of primitive cells outside thesite of colonization could be unimportant.

In an embodiment of the invention, one can use animal strains toleranttowards cells from antigenically different animals of the same ordifferent species. For example, nude mice, having no thymus-derivedcells, and SCID mice (Bosma et al, Ann. Rev. Imm. (1991) 9: 323-50),having low levels of both B-cells and T-cells, can be used asrecipients.

In another embodiment of the invention, one can use animal strains whichare from different species to provide donor cells (xenogeneic transfer).Donor cell-derived spermatozoa then may be mature along with endogenousspermatozoa in the epididymis. The egg of the foreign species wouldprovide the specificity of spermatozoal selection during in vitro or invivo fertilization. For example, if the donor is a chicken and therecipient male is a mouse, the spermatozoa collected from the mouse,when reintroduced into a fertile chicken would result in chickenprogeny. Chicken represent an important species for xenogeneic transferbecause they are economically an efficient source of quality protein andtesticular spermatozoa placed in the chicken oviduct are capable offertilizing the egg (Jones, et al., in "Oxford Reviews in ReproductionBiology," ed. Milligan, D. R. (Oxford University Press, England), 1993,vol. 15, pp 233-264).

Results of the introduction of the primitive cells are monitored afterrecovery of the males from the implantation procedure. As is known,depending on the species of the animal used, the period forspermatogenesis is approximately 30 to 60 days, and another 10 to 15days is needed for epididymal maturation of spermatozoa. See, e.g.,"Reproduction in Domestic Animals", 4th ed., Acad. Press (1991), ed. P.Cupps. Therefore, depending on the species used, males that havereceived primitive cells in accordance with the invention can beexamined beginning about two months after cell transfer.

The present invention is applicable to any species of animals, includinghuman, in which the male has testes, including but not limited totransgenics. The invention is also not limited to mammalian species. Itcan be used to provide animals and animal lines of many types with asingle, or many, novel genetic modification(s) or novelcharacteristic(s). The animals to which the present invention can beapplied include animals, such as rodents (e.g., mice, rats, etc.), birds(such as chickens and turkeys), wild and zoo species (such as zebras,lions, pandas, giraffes, polar bears, monkeys, sea otters, etc.) whichcan be modified to permit their use in cellular diagnosis or assays. Thepresent invention may also be advantageously applied to farm animalssuch as domesticated ruminants or fowl (e.g., cattle, chickens, turkeys,horses, swine, etc.), to imbue these animals with advantageous geneticmodification(s) or characteristic(s).

The donor and recipient animal can be the same animal patient. In thiscase, primitive cells are collected from the animal patient prior todestruction of the germ cell population and then reintroducedthereafter. This embodiment would preserve the ability of the animalpatient to reproduce following radiation therapy, for example which maybe necessary as a cancer treatment. Alternatively, spermatogonial stemcells may be harvested from the animal or patient and kept in culture orfrozen. Then when progeny are desired, the stem cells are transplantedto a recipient mouse testes. The donor animal or patient species eggcould then be fertilized by spermatozoa developed in the recipient mousetestes. There are no time constraints since the stem cells continuallyundergo self-renewal.

Once an initial fertilization event is achieved and the resultingoffspring is fertile, the animal line with its novel geneticmodification or characteristic is established, with the novel geneticmodification or characteristic being present in both male and femaleoffspring. Thus, in accordance with the invention one may produce ananimal harboring, in its testes only, a biologically functional germcell which is not native to that animal by repopulating its testicularseminiferous tubules. This (parent) animal can produce progeny. Everycell in the progeny is genetically non-native as compared to the parentanimal.

Both the parent animal and its progeny provided by the present inventionhave very varied uses, including uses in agriculture and biomedicine,including human gene therapy. An illustrative agricultural use of thepresent invention relates to increasing the breeding potential of avaluable stud animal. In this use, a testicular biopsy from a valuablestud animal is used to obtain stem cells for transfer into the treated(with either Busulfan®, radiation, etc.) testes of a recipient animal.The recipient animal thereafter produces sperm which are geneticallyfrom the valuable stud and natural matings with this recipient maleprovide an alternative to artificial insemination with ejaculates fromthe valuable stud. This illustrative technique is particularly useful asinsurance against an illness, injury or the death of a valuable studanimal. Likewise, spermatogonial stem cells of valuable animals may beharvested by biopsy from the testes and expanded in a mouse testes or inculture and subsequently frozen. The spermatogonial transplantationinvention provides for immortalization of the genetic composition(original or modified) of any male individuals of any species. Thisability will revolutionize the biology of inheritance.

Another illustrative application of the present invention is itsapplication to create (chimeric) animals useful in either biomedicine oragriculture. The present invention provides an advantageouscomplementation to existing transgenic techniques.

Existing transgenic techniques, when applied to animals other thanlaboratory mice, are often hampered by the difficulty of recoveringembryos, differing characteristics in embryos of different species, alack of knowledge about the specifics of reproductive timing in thegiven species, the economics of current techniques, etc. The presentinvention permits the difficulty and expense of embryological transgenicwork to be by-passed. In accordance with the present invention,spermatogonia stem cells can be genetically modified and thentransferred to recipient testes. The valuable genetic traits present inthe resultant germ cells can be passed onto the (transgenic) progeny ofthe recipient stud. This particular application of the present inventionis particularly important to the genetic engineering of largeagricultural animals.

The present invention also has applications in gene therapy, includinghuman gene therapy. For example, a patient with a deleterious genetictrait could undergo a testicular biopsy. Isolated stem cells can begenetically modified to correct the deleterious trait. The patient thenundergoes a treatment to remove the remaining germ cells from histestes, for example by specific irradiation of the testes. His testes(now devoid of germ cells) can then be recolonized by his own,genetically-corrected, stem cells. The patient can then father progenyfree from the worry that he would pass on a genetic disease to hisprogeny. Alternatively, the stem cells with the corrected gene could betransplanted to a mouse and the resulting sperm used for fertilizingeggs, thereby foregoing the use for reimplanting stem cells into theoriginal human testis.

Conventional cell markers, such as surface antigens or internal enzymes,can be employed in the transferred primitive cells to facilitatedetecting their presence in biopsy specimens of the testes. The presencein the ejaculate of spermatozoa with the characteristics of the markeris a reliable indication of success. Thus, in accordance with apreferred embodiment, at least one genetic marker is preferably used todistinguish the introduced cells from residual sperm that might arisefrom the host male. For example, transgenic mice strains that canproduce a characteristic stain in sperm cells which serve as donormarker cells can be used. The promoter from a gene active in developingsperm cells (Zfy-1 or a homolog) is used to drive expression of the genefor the bacterial enzyme beta-galactosidase (lac Z) in transgenic mice.In the presence of the reagent X-gal, sperm cells from these transgenicmice stain blue, unlike those from normal mice. Thus, when acting asdonors, the cells can be easily distinguished from host cells, servingas a marker for analysis.

Co-insertion of both a gene of interest and a marker gene into stemcells to be used as donors may be carried out in accordance with thefollowing illustration. The Zfy-1/beta-galactosidase construct describedabove is mixed in solution with a genetic construct of interest toeither co-inject into eggs to produce transgenic mice to provideeventual donor cells or transfect into tissue culture cells to be usedas donors. A proportion of the resulting animals or cell lines willcontain both constructs, providing donor cells with an easily-detectiblemarker as well as the gene of interest.

Since colonization usually only takes place in some tubules, low numbersof spermatozoa can result. However, any male in which some transferredprimitive cells have developed into mature spermatozoa is useful inaccordance with the invention since a variety of conventional techniquesexist to achieve fertilization in animals with low sperm numbers. Thesetechniques include, but are not limited to: hormonal treatment,abstinence, artificial insemination, in vitro fertilization, zonadrilling, and microintroduction of sperm into the egg.

The experiments described below, which are provided for purposes ofnon-limiting illustration of some embodiments of the invention,demonstrate the survival, differentiation and development to enterstages of spermatogenesis of cells transferred in accordance with theinvention and the cross-strain utility of the invention, and therebyprovide strong evidence of the cross-species potential of the presentinvention.

EXAMPLES Example A

Introduction

Foreign potential stem cells for transfer were isolated from the testesof very young male mice, usually between 3 to 10 days of age. Thesecells carried a reporter or marker gene encoding the E. coliβ-galactosidase (lacZ) gene. This gene is not normally present in themouse genome. However, if a cell contains the gene, it will make theenzyme β-galactosidase. In the presence of the reagent X-gal, the cellwill then stain blue. In addition, the transgene in these cells willonly be active in late stages of spermatogenesis, in the round spermatidand later stages, because the lacZ structural gene is under control of apromoter or activating DNA sequence designated ZF. These late stages ofspermatogenesis are not present in the neonatal testes. Therefore thetransgene is not active in neonatal testes and these cells cannot bestained blue nor do any of the cells have the appearance of maturespermatozoa at this early age. As a result of this experimentalprocedure, the transferred cells must not only survive, but they mustundergo differentiation and development to become late stages ofspermatogenesis in order to stain blue. Furthermore, the transferredcell descendants can easily be distinguished from any endogenous spermcells of the recipient host mouse (should any be present), because theseendogenous cells will not stain blue.

An example of a cross section of a seminiferous tubule from the testisof an adult control mouse not containing the transgene and treated withX-gal is shown in FIG. 1. There are no cells that show blue or darkstaining. In contrast, a cross section of a seminiferous tubule from thetestis of an adult mouse containing the lacZ transgene and treated withX-gal is shown in FIG. 2. There is intense blue (dark) staining in allthe mature stages of spermatogenesis, which occupy the center of thetubule. FIG. 1 and 2 represent examples of a negative and positivecontrol, respectively. They are referred to below in the experimentaldescriptions.

The cells for transfer were obtained from neonatal mice (3 to 10 daysold) at a time before they would show any stain because of theirimmature stage of differentiation. These cells are reporter cells andcarry the transgene ZF-lacZ; they are obtained from the testes of hybridmice that carry antigenic determinants from C57BL/6 and SJL mice. Thecells were then injected into the seminiferous tubules of recipient orhost mice at several sites on the testicular surface.

The recipient mice were of several types as follows:

1. Hybrid mice of C57BL/6 x SJL parent stock. These mice wereimmunologically tolerant of the donor cells and should not reject them.The host mice were prepared by treatment with Busulfan® to destroy theirendogenous sperm cells.

2. Mice that carry the W-mutation. Homozygotes and compoundheterozygotes for certain alleles of this gene are completely devoid ofsperm cells in the adult. Therefore, any sperm cells in theirseminiferous tubules must come from the differentiation of transferredcells. Furthermore, these mice are immunologically incompatible with thedonor cell strain of mice because the W-mice are of C57 background andwould not be tolerant of the SJL antigens on the surface of cellscontaining the ZF-lacZ transgene.

3. Inbred mice of the 129/SV strain. These mice are immunologicallyincompatible with the donor cells because the cells contain C57BL/6 andSJL antigens and 129/SV mice would recognize both strains as foreign.Foreign cells would be rejected and destroyed by a mouse if present in anormal environment, such as a skin or organ graft. However, some partsof the testis, particularly the inner region of the seminiferoustubules, is considered to have a degree of immunological privilege (i.e.They do not reject foreign tissue as readily as other body locations.The uterus is the best example of an immunologically privilegedlocation, since it does not reject the fetus carrying the maleantigens).

The implementation and use of the invention has substantiated in thefollowing examples, and the results obtained provided proof of severalimportant aspects of the invention. The examples describingimplementation of the invention are as follows.

Experimental

1. Primitive stem cells or spermatogonia can be transferred from oneanimal into another host animal and the donor cells will survive, divideand differentiate. Cells were taken from neonatal testes of hybrid mice(C57BL/6 x SJL) containing the ZF-lacZ transgene, and these cells weremicroinjected into the seminiferous tubules of Busulfan® treated hybrid(C57BL/6 x SJL) mice. The host testes were removed at various times(days to months) following the injection and examined for the presenceof cells from the donor animal by analyzing the testes for the presenceof lacZ staining. A number of animals were found that clearlydemonstrated the success of the transfer as well as survival, divisionand differentiation of the donor cells. In FIG. 3A is shown a crosssection from one of these testes, and it is clear that a number oftubules are stained blue (dark). This is the same type staining seen inFIG. 2 and, therefore, represents descendants of donor cells rather thanregeneration of endogenous cells, which would not stain but would looklike FIG. 1. Furthermore, mature spermatozoa are seen in some tubules.FIG. 3B is a higher magnification of one of the tubules from FIG. 3A.The mature spermatozoa are clearly seen as thin dark nuclei toward thecenter of the tubule. The blue stain (dark) is very obvious at thismagnification. Survival, division and differentiation of donor cellsmust take place to achieve this result. Donor cells will not stainbecause they are immature; endogenous cells (should any regenerate) willnot stain because they lack the transgene; and no mature spermatozoawere present among the donor cells. A few transferred stem cells dividedand differentiated to fill this seminiferous tubule in the host animal.

2. In the second implementation of the invention, the same type cellsdescribed above, from hybrid mice and carrying the ZF-lacZ transgene,were transferred into the seminiferous tubules of W-mutant mice. Thesemice are devoid of endogenous sperm cells. The success of the procedureis demonstrated in FIG. 3C which shows a cross section of a tubule thatreceived donor cells. There is blue staining of cells in this crosssection demonstrating their derivation from donor cells. Furthermore,this mutant animal is incapable of generating sperm cells. In thisembodiment of the invention the donor cells have colonized a completelysterile testis in an animal that is not immunologically tolerant of thedonor mouse strain. The testis environment has provided an immunologicalprotection as anticipated in the invention application. The examinationof this testis was performed 120 days after donor cell transferindicating that cell survival, division and differentiation continues inthe host for a long period, probably until death.

3. In the third implementation of the invention, the same type donorcell was transferred into the seminiferous tubule of a 129/SV inbredmouse. The mouse had previously been treated with Busulfan® to destroyendogenous sperm cells. The result is shown in FIG. 3D. Again the donorcells (stained blue or dark) have colonized this tubule. Thedifferentiation has not proceeded as far as in the examples above;perhaps, because the transfers were among the first performed whileimprovements were still underway. However, this example demonstratestolerance of foreign cells in the tubule despite very strongimmunological differences. The examination of this testis was performed110 days after donor cell transfer, indicating a very long period ofdonor cell survival.

Example B Material and Methods

Cell Preparation

Male germ cells were isolated from mice of three different ages: betweenfetal (f) day 18 and postnatal (pn) day 2; between pn days 5 and 15; andbetween pn days 21 and 28. The basic procedures used have been described(Bellve' et al., (1977) J. Cell Biol. 74, 68-85). Several modificationswere introduced to increase the number and improve the handling ofisolated cells. Briefly, 12 to 40 testes were collected (the largernumbers from the youngest animals), the tunica removed, and the exposedtubules subjected to collagenase (1 mg/ml) treatment followed by trypsin(0.25%) digestion. The released cells were centrifuged at 600×G at 16°for 5 minutes. Following centrifugation, the supernatant was removed andthe cells resuspended in injection medium. The composition of the mediumwas based on a formula originally developed for mouse eggs (Brinster, R.L. (1965) J. Reprod. Fert. 10, 227-240) and subsequently modified foruse with primordial germ cells (Brinster et al., (1977) Exptl. Cell Res.109, 111-117). These cell types have a requirement or preference forpyruvate and lactate as energy substrates, and male germ cells may sharethis characteristic because of their origin from primordial germ cells.The composition of the injection medium was: 132 mM NaCl, 7.8 mM Na₂HPO₄, 2.6 mM KCl, 1.1 mM KH₂ PO₄, 0.1 mM EDTA, 0.25 mM pyruvate, 3 mMlactate, 1 mM glutamine, 5.5 mM glucose, 5 mg/ml bovine serum albumin,200 μg/ml DNAse I, pH 7.4. The cells were resuspended in 1.6 ml of theinjection medium, and 400 μl allocated to microcentrifuge tubes on ice.Cell concentration values for different age testes were determined onsamples of cells collected in an identical manner on days wheninjections were not performed. There was variation in cell concentrationfrom day to day resulting from the number of testes available and theage of the donor males. An attempt was made to maximize the number ofcells available in order to inject a concentrated solution, since thenumber of stem cells is unknown but thought to be small. One tubecontaining 400 μl was used to inject the testes of a single mouse.Before introducing the cell suspension into the injection pipette, 50 μlof filtered trypan blue (4%) was added to the tube and the suspensionagitated. The dye allowed visualization of solution flow in theseminiferous tubule and was used to determine the number of dead cellsin the injection suspension, which was generally less than 5 percent. Itwas not possible to measure the volume of cell suspension that enteredthe tubules, but the percent of surface tubules that filled withsolution was recorded for each recipient testis.

Sertoli cells were isolated and maintained according to methodspreviously described (Karl et al., (1990) Methods In Enzymology 190,71-75). They were added to the germ cells just before injection into thetubules. Embryonic stem cells (AB-1) were a gift from Alan Bradley andwere maintained as previously described (Bradley, et al., (1992)Bio/Technology, 10, 534-539; Robertson, E. J. (1987) in"Teratocarcinomas and Embryonic Stem Cells: A Practical Approach", ed.Robertson, E. J. (MI Press Limited, Oxford, England), pp. 71-112).

Transplantation Procedure

Two separate protocols were used to transfer donor cells into recipientmice. In protocol 1, donor cells were isolated from the testes ofC57BL/6 mice that were homozygous for a dominant mutant that resulted intan belly hair. These cells were transferred into testes of compoundheterozygous or homozygous mutant W mice (designated W/W) which do notshow spermatogenesis (reviewed in Silvers, W. K. (1979) The Coat Colorsof Mice: A Model for Mammalian Gene Action and Interaction.(Springer-Verlag, New York). pp. 206-241). In protocol 2, donor cellswere isolated from mice (designated ZFlacZ) that were heterozygous foran E. coli β-galactosidase transgene, which allowed round spermatids tobe stained blue following incubation with substrate (Zambrowicz, et al.,(1994) Development 120, 1549-1559). Donor cells from these mice wereinjected into testes of (C57BL/6 x SJL) F1 males that had been treatedwith Busulfan® (40 mg/kg intraperitoneally) at 4 to 6 weeks of age. Thistreatment destroys spermatogenic stem cells (Bucci, et al., (1987)Mutation Research 176, 259-268).

Cell Injection

To minimize the number of injection sites and to increase the efficiencyof injection concentrated cell suspension, a glass pipette with a 1 mmoutside diameter and a 40μ tip was secured in a micropipette holder (WPICat. No. MPH6S). The tip of a 1 ml plastic syringe was then insertedinto the other end of the micropipette holder, and the syringe was cutat the 0.4 ml mark. The cell solution containing the dye was depositedin the syringe barrel, which was then screwed onto the metal end of anEppendorf capillary holder attached by tubing to a pressure injector(Eppendorf Model 5242). To expose the tubules, only 1 to 3 smallincisions (1-3 mm) were made in the tunica per testis. With thesemodifications, an average of 80 percent of the surface tubules could befilled with concentrated cell suspension (Table 1).

Analysis of Recipient Testes

To allow donor cells to undergo at least one cycle of spermatogenesis,recipient males were maintained a minimum of 50 days following injectionbefore sacrifice. In protocol 1, the testes were fixed and 5μmicroscopic sections cut and stained with hemotoxin and eosin. Thenumber of tubule cross sections showing any stages of spermatogenesiswas recorded. In protocol 2, the testes were incubated with5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) and the number oftubules that stained blue recorded, up to a maximum of 12; beyond this,it was difficult to identify individual tubules because of theirtortuous course, and these testes were recorded as 12+. For some mice inprotocol 2, microscopic sections were cut and stained with neutral fastred. The number of tubule cross sections that stained blue generally wasgreater than the number of tubules visibly blue on gross examination.Progeny of recipient males (protocol 2 only) were analyzed by incubatingtestes with X-gal or by assaying for the presence of the transgene(Zambrowicz, et al., (1994) Development 120, 1549-1559).

Results

Donor cells were collected from mouse testes at 3 representativedevelopmental stages. The first, between fetal day 18 and pn day 2,provided primarily gonocytes and mitotically active Sertoli cells. Thesecond, between pn days 5 and 15, provided germ cells and Sertoli cellsin transition between the perinatal and adult states. The third, betweenpn days 21 and 28, provided quiescent Sertoli cells and a germ celldistribution approaching that of mature testes. The average time fromcell transplantation to analysis was approximately 90 days, with a rangeof 54 to 300 days (Table 1). This represented between 2 and 10spermatogenic cycles. In total, 245 testes were injected with donorcells, of which 173 (71%) showed evidence that transplanted cells hadsurvived, colonized recipient seminiferous tubules, and initiatedspermatogenesis (Table 1). Cells isolated from perinatal testes (18 f to2 pn) colonized 24 of 86 injected tests, with an average of 3 to 4tubules (out of approximately 150 to 300 visible in a cross section)showing evidence of donor cell spermatogenesis. Donor cells isolatedfrom pn day 5 to 15 and pn day 21 to 28 displayed a greater ability tocolonize recipient testes: 90 of 96 and 59 of 63 testes, respectively,showed evidence of donor cell spermatogenesis. Donor cell age and strain(protocol 1 vs 2) both had a significant effect on the number of testescolonized (Table 1). The number of tubules supporting spermatogenesisfor the pn day 5 to 15 and 21 to 28 cells was also significantly greaterthan for the perinatal cells. Although the concentration of donor cellsinjected was lower for suspensions from perinatal animals, reflectingthe small size of donor testes, the percent of surface tubules filledwith cell suspension (˜80%) and the percent live cells, as indicated bytrypan blue, were similar for all 3 donor stage cell preparations.

                                      TABLE 1                                     __________________________________________________________________________    Spermatogonial Stem Cell Transplantation into Recipient Testes                Donor Cell                Parent                                              Age*  Concentration of                                                                        Donor Cell                                                                          Testes                                                                            Surface Area                                                                        Time to                                                                            Tests with                                                                          Tubules with                       (Days)                                                                              Donor Cells.sup.+  (× 10.sup.6)                                                   Strain.sup.‡                                                             Injected                                                                          Covered.sup.§                                                                  Analysis.sup.¶                                                           Donor Cells.sup.∥                                                          Donor Cells**                      __________________________________________________________________________    18f-2pn                                                                             35 ± 16                                                                              C57BL/6                                                                             46  78 ± 20                                                                          81 ± 20                                                                         6 (13)                                                                              3.5 ± 2.3                              10-51!              30-100!                                                                             54-130!    1-7!                                              ZFlacZ                                                                              40  73 ± 21                                                                          81+14                                                                              18 (45)                                                                             3.2 ± 2.5                                                  40-100!                                                                             60-110!    1-9!                              5pn-15pn                                                                            71 ± 18                                                                              C57BL/6                                                                             47  80 ± 19                                                                          117 ± 64                                                                        41 (87)                                                                             14.5 ± 16.2                            44-108!             30-100!                                                                             70-300!    1-70!                                             ZFlacZ                                                                              49  80 ± 15                                                                          87 ± 37                                                                          49 (100)                                                                           8.1 ± 4.0                                                  50-100!                                                                             60-197!    1-12+!                            21pn-28pn                                                                           83 ± 32                                                                              C57BL/6                                                                             11  86 ± 7                                                                           99 ± 43                                                                         7 (64)                                                                              16.7 ± 6.4                             45-123!             75-95!                                                                              70-180!    9-26!                                             ZFlacZ                                                                              52  83 ± 15                                                                          80 ± 25                                                                          52 (100)                                                                           5.1 ± 3.4                                                  40-100!                                                                             60-155!    1-12+!                            __________________________________________________________________________     Table 1 are: Mean ± S.D.; Range is in brackets,                            *Donor cell mice of three ages provided testis cells for transfer into        recipients. f, fetal; pn, post natal.                                         .sup.+ Number of determinations of cell concentration was 6 for 18f-2pn,      for 5pn-15pn, 6 for 21pn-28pn.                                                .sup.‡ C57BL/6 testis cells were transferred to the                seminiferous tubules of homozygous or compound heterozygous W/W mice that     lack spermatogenesis (protocol 1). ZFlacZ testis cells from mice of           C57BL/6 × SJL genotype were transferred to the seminiferous tubules     of (C57BL/6 × SJL) F1 hybrid mice in which endogenous                   spermatogenesis had been destroyed by Busulfan ® treatment (protocol      2). ZFlacZ mice were heterozygous for a transgene (-3.5 kb cEMS177/lacZ),     and the transgenic lines were designated TgN(c177lacZ)226Bri and              TgN(c177lacZ)227Bri (Zambrowicz, et al., (1994) Development 120,              1549-1559).                                                                   .sup.§ Percent of the surface seminiferous tubules in the recipient      testis filled by the injected cell suspension.                                .sup.¶ Number of days from injection of donor cells to analysis     of the recipient testis for presence of spermatogenesis. This represents      the time available for donor cell proliferation.                              .sup.∥ Effect of donor cell age and donor cell strain (C57BL/6 v     ZFlacZ) were both significant by analysis of variance (0.05 > P > 0.01).      **Number of tubules with spermatogenesis in those recipient testes that       had evidence of donor cell colonization. In C57BL/6 donor cell recipients     the number of tubule cross sections with evidence of spermatogenesis were     counted. In ZFlacZ donor cell recipients, the number of individual tubule     stained, blue were counted up to a maximum of 12. Tubules in testes with      more than 12 stained could not be accurately counted. Therefore, in this      column the values for C57BL/6 cells are not directly comparable tpo those     for ZFlacZ cells. The difference between the C57BL/6 values for the           18f-2pn age group and the other two groups was significant (P < 0.001).       The difference between the ZFlacZ values for the 18f-2pn age group and th     5pn-15pn age group was significant (P < 0.001), and the difference betwee     the 18f-2pn age group and the 21pn-28 pn age group was also significant       (0.02 > P > 0.01). Analyses by ttest. The ZFlacZ statistical comparisons      underestimate the true difference, because in testes with 12+ tubules         staining the exact number of tubules with spermatogenesis is too great to     count accurately.                                                        

Following colonization of recipient seminiferous tubules, donor cellsgenerally established a morphological pattern representative of normalspermatogenesis. For protocol 1, all stages of sperm celldifferentiation could be identified in microscopic cross sections of theseminiferous tubules. None of these stages was seen in control testesnot injected with cells, because there is no endogenous spermatogenesisin W mice, or in tubules of injected testes not populated with donorcells. Likewise, ZFlacZ donor cells (protocol 2) established normalspermatogenesis in Busulfan®-treated recipients with typical sperm cellassociations and morphology of the tubules, and mature spermatozoa wereproduced (FIG. 4A). The testes of mice injected with Busulfan® but notreceiving donor cells do not generally have tubules with spermatogenesis(Bucci, et al., (1987) Mutation Research 176, 259-268). In micereceiving a lower dose of Busulfan® (e.g., if there was leakage at thetime of injection) some tubules could be repopulated by survivingendogenous stem cells. However, these tubules would be distinct, sinceround spermatids and subsequent stages derived from endogenous stemcells do not stain blue with X-gal. The success of the transferred donorcells in colonizing the recipient testis varied, and some testes fromZFlacZ mice demonstrated up to 100 cross sections containing variousstages of spermatogenesis. In testes with the most extensivecolonization, by either C57BL/6 or ZFlacZ donor cells, maturespermatozoa were transported to the epididymis (FIG. 4B). No sperm wereseen in epididymal tubules when recipient testes were not colonized bydonor cells.

As anticipated based upon the observation of spermatozoa in theepididymides of some experimental mice, spermatozoa could also beidentified in the ejaculate of these recipient males. Initially, thenumber of spermatozoa was insufficient to result in progeny. An approachto produce fertile production of offspring from donor-derivedspermatozoa would be to provide carrier spermatozoa. As a consequence ofoccasional Busulfan® leakage at the time of injection or variability ofindividual animal susceptibility, a few experimental recipients mayacquire fertility following tubule repopulation by endogenous stemcells. If donor cells have also become established, a fraction oftubules with spermatogenesis will stain blue (FIG. 5A, B), and theejaculate will contain spermatozoa derived from endogenous andtransplanted stem cells. One-half the male progeny that develop fromeggs fertilized with spermatozoa of ZFlacZ donor stem cell origin shouldhave testes that stain blue with X-gal, because transferred ZFlacZ cellsare heterozygous for the transgene. Testes from progeny of a recipientin which spermatogenesis occurred from endogenous and transplanted stemcells are shown in FIG. 6; however, only one of 122 male offspringcarried the transgene. To specifically generate recipients that wouldreestablish endogenous spermatogenesis, protocol 2 was used; ten maleswere treated with 30 mg/kg Busulfan®, and the testes injected with donorcells from pn day 5 to 15 or 21 to 28 mice. Testes of seven wereexamined 65 to 290 days after injection. All were enlarged relative toanimals treated with 40 mg/kg Busulfan®, indicating a significant levelof spermatogenesis. The testes contained 8.2±4.0; (mean±standarddeviation M±SD) blue staining tubules. Of the 3 remaining males, onethat received pn day 5 to 15 cells is still infertile, one that receivedpn day 21 to 28 cells has sired 6 of 39 transgenic progeny, and thethird that receiving pn day 5 to 15 cells has sired 6 of 15 transgenicprogeny. Offspring were born beginning approximately 8 months afterdonor cell injection. With this approach, up to 80 percent of theprogeny of the male with the most successful colonization are fromdonor-derived spermatozoa, half of which carry the transgene.

Although injected cell suspension contained donor Sertoli cells, severalexperiments were performed in which Sertoli cells (10⁵ to 10⁷) isolatedas described were added to the donor cell suspension to determine ifthis would enhance repopulation. Using protocol 1 and donor cells frompn day 5 to 15 mice, 10 to 12 recipient testes demonstratedspermatogenesis, with 13.0±12.7 (M±SD) tubule cross sections colonizedper testis. With protocol 2 and the same age donor cells, 24 of 25testes had stained tubules with 9.0±3.7 (M±SD) tubules stained pertestis. The average time of analysis of the recipients after donor cellinjection was 112 days. In general, the degree of colonization with andwithout additional Sertoli cells was similar (compare to Table 1).

Discussion

The studies described above demonstrate that stem cells can be harvestedfrom donor testes, maintained in vitro, transferred to a recipienttestis, establish normal spermatogenesis, and produce functionalspermatozoa that fertilize eggs and result in offspring. The productionof young from the transplanted spermatogonial stem cells and the highpercent of recipient spermatozoa arising from the transplanted cellsindicates that colonization of the seminiferous tubules can be veryeffective. The procedure used in the above example showndiagrammatically in FIG. 7.

In these studies, donor cell populations were mixed, containing Sertolicells and various stages of differentiating sperm cells that varied withthe age of the donor testes (Bellve', A. R. (1979) in Oxford Reviews ofReproductive Biology, ed. Finn, C. A. (Clarendon Press, Oxford, England)Vol. 1, pp. 159-261; de Kretser, et al (1998) in The Physiology ofReproduction, eds. Knobil, E. & Neill, J. (Raven Press, Ltd., New York)pp. 837-932; Bellve' et al., (1977) J. Cell Biol. 74, 68-85). Among thisdiverse group of cell types, only cells with stem cell potential couldinitiate spermatogenesis. Spermatocytes, meiotic stages, and spermatidsare not capable of self-renewal. Thus, even if donor cells representinglater stages of spermatogonia established an interaction with recipientSertoli cells and continued to differentiate, they would become fullymature and be shed into the lumen by 35 days post-transfer (de Kretser,et al (1998) in The Physiology of Reproduction, eds. Knobil, E. & Neill,J. (Raven Press, Ltd., New York, N.Y.) pp. 837-932; Russell, et al.,(1990) in Histological and Histopathological Evaluation of the Testis,(Cache River Press, Clearwater, Fla.) pp. 1-40). Spermatogenic elementsvisible in recipient testes after this time would necessarily havearisen from donor cells with stem cell potential.

The age of the testes from which donor cells were isolated had aninfluence on the efficiency of colonization of recipient testes. Atbirth the germ cells are gonocytes, but during the first week of lifethese cells differentiate into spermatogonia, which divide and begin todifferentiate (Bellve', A. R. (1979) in Oxford Reviews of ReproductiveBiology, ed. Finn, C. A. (Clarendon Press, Oxford, England) Vol. 1, pp.159-261; de Kretser, et al (1998) in The Physiology of Reproduction,eds. Knobil, E. & Neill, J. (Raven Press, Ltd., New York) pp. 837-932).Thereafter, the spermatogonial cell population expands anddifferentiating stages of spermatogenesis appear successively. Inprepubertal mice this first wave of spermatogenesis is synchronized,with spermatocytes appearing at about day 10, spermatids at about day20, and mature spermatozoa at about day 35 (de Kretser, et al (1998) inThe Physiology of Reproduction, eds. Knobil, E. & Neill, J. (RavenPress, Ltd., New York) pp. 837-932; Bellve' et al., (1977) J. Cell Biol.74, 68-85). Sertoli cells also undergo changes during this period. Theycontinue to divide until between 10 and 14 days after birth, after whichthey become mitotically quiescent (Gondos, et al., (1993) in The SertoliCell, eds. Russell, L. D. & Griswold, M. D. (Cache River Press,Clearwater, Fla.) pp. 116-154). Thus, by recovering donor cells atbirth, pn day 5 to 15, and pn day 21 to 28, three different mixed cellpopulations were tested for stem cell potential. Surprisingly, theresults suggest that gonocytes, despite their primitive stage ofdevelopment, were less efficient as donor cells than older populationsof cells containing spermatogonia. However, it has not been determinedwhether the stem cell populations in the 3 developmental stages testedhave a differential sensitivity to the harvesting protocol employed.Although the number of dead cells as indicated by trypan blue stainingwas similar for the 3 cell preparations, differential killing of smallpopulations of stem cells may not have been detectable. Also, stem cellsare likely to make up a different fraction of the total cell populationat each stage (Bellve', A. R. (1979) in Oxford Reviews of ReproductiveBiology, ed. Finn, C. A. (Clarendon Press, Oxford, England) Vol. 1, pp.159-261; de Kretser, et al (1998) in The Physiology of Reproduction,eds. Knobil, E. & Neill, J. (Raven Press, Ltd., New York) pp. 837-932;Russell, et al., (1990) in Histological and Histopathological Evaluationof the Testis, (Cache River Press, Clearwater, Fla.) pp. 1-40.). Thesefactors may also account for the lower efficiency of C57BL/6 donor cellsin colonizing recipients. However, an additional element in this effectcould be an inferior ability of the tubules in W recipients to supportspermatogenesis.

Spermatogenesis was morphologically normal in many tubules examined(Russell, et al., (1990) in Histological and HistopathologicalEvaluation of the Testis, (Cache River Press, Clearwater, Fla.) pp.1-40), indicating effective colonization of seminiferous tubules. Thedonor cell population contained both germ cells and Sertoli cells;therefore, it has not yet been determined whether the interactingSertoli-spermatogonial units that established a spermatogenic colony andrepopulated the tubule were composed of donor germ cells interactingwith endogenous Sertoli cells, with transferred Sertoli cells, or both.However, when additional Sertoli cells were coinjected with germ cellsthey did not have a dramatic effect on colonization efficiency. Thissuggests that Sertoli cells were not a limiting factor, and may indicatethat endogenous Sertoli cells were adequate to support recolonization.

The most striking result of these experiments was production ofoffspring from donor cell-derived spermatozoa.

Example C Reconstitution of Spermatogenesis from Frozen SpermatogonialStem Cells

Spermatozoa from a number of species can be cryopreserved and thensubsequently used to fertilize eggs¹. However, this technique hasseveral limitations. First, the freezing protocol varies for eachspecies and must be determined empirically, and for some speciesappropriate methods have not yet been identified¹,2. Second, since thesecells are fully differentiated, they will not undergo replication whenthawed, and recombination of genetic information cannot occur. Thefollowing example demonstrates that, by using the recently developedspermatogonial transplantation technique³,4, male germ line stem cellscan be successfully cryopreserved. Donor testis cells isolated fromprepubertal or adult animals and frozen for 4 to 156 days at -196° C.were able to generate spermatogenesis in recipient seminiferous tubules.Relatively standard preservation techniques were used, suggesting thatmale germ cells from other species can also be stored for long periods.Because transplanted testis stem cells will ultimately undergoreplication and meiotic recombination during spermatogenesis, one mightconsider these preserved male germ lines as biologically immortal.

After birth in mammals, female germ line cells do not undergoreplication, and their number decreases with age⁵. However, in the male,germ line cell division and spermatogenesis continues throughout most orall of adult life. Spermatogenesis is complex, highly ordered and veryproductive⁶,7. At the foundation of this process is the spermatogonialstem cell, which both self-renews and provides a population of cellsthat increases in number and differentiates to form maturespermatozoa⁶⁻⁸. In most mammals the differentiation process is completedover the course of 30 to 60 days, and the population of differentiatingcells increases dramatically. A single rat stem cell is capable ofproducing 4096 mature spermatozoa⁶. However, the efficiency is never100%, and some cellular degeneration typically occurs at eachdifferentiation step⁶,7.

Surprisingly, there are few reports of efforts to cryopreserve male germline stem cells. Early attempts to freeze testicular tissue met withonly limited success (reviewed in 9, 10). However, in one study,cytological evidence of spermatogenesis was observed followingtransplantation of frozen and thawed immature rat testis pieces to thescrotum of a castrated adult¹⁰.

To determine if male germ cells could be cryopreserved in suspension,testis cells were collected from prepubertal or adult mice carrying alacZ transgene that allows round spermatids and cells in later steps ofspermiogenesis to be stained blue when incubated with X-gal (FIG.8A)³,4,11. This provides a useful marker of testis cells, becausesomatic cells do not stain and a single spermatogonial stem cell expandsinto a large clone of round spermatids (ref. 6) making identification ofstem cell progeny easy and unequivocal³,4. Donor testis cells werecollected and frozen slowly at -70° C., then subsequently stored forvarying periods of time at -196° C., using techniques similar to thosegenerally employed for somatic cells¹². The cells were then thawed andtransplanted into the seminiferous tubules of recipient mice in whichendogenous spermatogenesis had been destroyed by busulfan treatment(FIG. 8B)³,4,13 . Following transfer to recipient tubules, these donorcells established spermatogenesis. Areas of the recipient testespopulated by cryopreserved donor cells could be readily identified byblue staining (FIG. 8C, D). Donor cells from both adult (FIG. 8C) andprepubertal (FIG. 8D) animals were effective in colonizing recipienttestes. Areas not repopulated by donor cells will not stain blue³,4.

To assess the morphological fidelity of donor cell spermatogenesis,microscopic sections of recipient seminiferous tubules were examined. Indonor testes, all spermatogenic stages from round spermatids to matureelongating spermatids were stained blue (FIG. 9A). In busulfan-treatedrecipient testes, which did not receive transplanted donor cells, theseminiferous tubules lacked germ cell stages and contained only Sertolicells lining the tubule (FIG. 9B). However, following transplantation ofdonor cells, blue-stained tubules contained germ cell elements fromround spermatids to mature elongating spermatids (FIG. 9C, D). Thestructure and pattern of spermatogenesis resulting from donor cellsresembled that seen in control testes (FIG. 9A), and was similar fordonor cells from adult (FIG. 9C) and prepubertal (FIG. 9D) mice.Previous studies have demonstrated that spermatozoa produced fromtransplanted testis cells can fertilize eggs, which subsequently developinto normal offspring⁴.

Four separate experiments, in which testis cells were frozen for between4 and 156 days, are summarized in Table 2. In each experiment, cellsstored at -196° C. were able to repopulate recipient testes and generatenormal spermatogenesis in recipients. In total, 22 of 30 (73%) recipienttestes showed spermatogenesis from transplanted cells. In successfullyreconstituted testes, from 1 to more than 12 seminiferous tubules werecolonized. Because of intertwining among the convoluted tubules⁶,7,14,it was not possible to accurately count greater than 12 stained tubulesin a single testis. The overall rate of successful colonization was notdecreased by the time at -196° C., since in the least successfulexperiment the cells were frozen only 4 days. Variation in colonizationof recipient testes by donor cells is generally correlated with thenumber of cells microinjected and the surface area of the testis covered(ref 4 and unpublished). The later parameter provides an estimate of thedegree of filling of the seminiferous tubules in the testis.

Successful cryopreservation of spermatogonial stem cells by routineprocedures is surprising considering the difficulty that has beenassociated with freezing mature spermatozoa¹,2. Indeed, it has beendifficult to establish a simple, reliable cryopreservation system formouse spermatozoa², although the dramatic increase in the number oftransgenic mouse lines has stimulated considerable effort in this area.Likely, the reason for successful spermatogonial stem cellcryopreservation results from the disparate morphologic characteristicsof the reproductive cell populations⁶. During the differentiationprocess from spermatogonia to spermatozoa, there are major structuralmodifications, including the loss of cytoplasm and restructuring ofnuclear DNA¹⁵,16, which may render spermatozoa more sensitive tofreezing and thawing. The stem cell appears able to respond to freezingmuch like a typical somatic cell, and recover with full functionalcapability. Given the apparent morphological similarity amongspermatogonia of various species⁶,14,17, it appears that the stem cellsof most or all mammalian species can be stored indefinitely at -196° C.,then subsequently used to generate functional spermatogenesis and maturespermatozoa in the appropriate seminiferous tubule microenvironment. Atestis of the species of donor cell origin will likely provide the mostcompatible site for regeneration of spermatogenesis, yet there isevidence that testes of other species may also be suitable as asurrogate host¹⁸.

In these experiments, recipient males were maintained only long enoughto generate spermatogenesis and mature spermatozoa from transplantedcells. Previous work has shown that donor cell derived spermatozoa canfertilize eggs effectively. In those studies, 80 percent of the progenyfrom one recipient male were shown to carry the donor haplotype⁴. Whilethis high level of colonization can be achieved, it is not essential toperpetuate the germ line, because intracytoplasmic injection of a singlespermatozoa into the oocyte will result in fertilization and birth oflive young in mice and humans¹⁹,20. Furthermore, the recentdemonstration that intracytoplasmic injection of a round spermatid canproduce young, means that spermatogenesis in the recipient must onlyproceed two-thirds of the way to completion²¹,22. The complicatedprocess of spermatozoa shaping, from round spermatid to maturespermatozoa, that occupies the last one-third of the differentiationpathway is not required if the haploid male germ cell nucleus isinjected into the oocyte¹⁹⁻²².

Cryopreservation of spermatogonial stem cells is likely to havefar-reaching consequences. Testis cells from unique experimental orlivestock animals of any age, from prepuberty to maturity, can now befrozen with the expectation that they will generate spermatogenesis at alater date. The germ line of the animal could then be reclaimed whenneeded. In addition, identifying the conditions necessary to culture andmodify spermatogonial stem cells prior to or after freezing willdramatically increase the value of these cells. Thus, this technique isfar different than cryopreservation of mature spermatozoa, for which theprotocol must be determined for each species and which then preservesonly a static cell population no longer able to undergo geneticrecombination or proliferation.

One must now begin to address the medical issue of whether males likelyto loose germ cells (e.g. following chemotherapy) should have testicularbiopsy material cryopreserved for possible reintroduction followingcessation of treatment. In addition, males with azoospermia may bebenefitted by this technique. Although mature spermatozoa are oftenabsent in these individuals, or few in number, the stem cells present inthe testes may be capable of generating spermatogenesis in a surrogate.Ideally testis cells could be cryopreserved and periodically expanded innumber when appropriate techniques are developed for introduction into atestis. The recent demonstration that rat spermatogenesis will proceednormally in the seminiferous tubules of an immunodeficient mousesuggests that xenogeneic spermatogonial transplantation may be possiblefor other species, and thus facilitate the identification ofsurrogates¹⁸. Probably the widest use of cryopreservation will be forthe germ lines of valuable experimental males in research, agricultureanimals that die before puberty or are too old to breed yet havevaluable germ lines, and males from exotic or endangered species. Forexample, the genetic diversity of a species with a small number ofindividuals might be partially protected by cyropreservation oftesticular tissue from males of all ages. The possibility of preservinga male germ line indefinitely will be enormously valuable in veterinarymedicine as well as human medicine. An important aspect of the techniqueis that in biological terms cyropreservation of the germ lineeffectively establishes the potential of generating at any time clonesof the original male following spermatogonial transplantation tomultiple recipients. When the ability to culture these cells isachieved, the procedure of cryopreservation will have even greater use.The essence of biology is the proliferation and meiotic recombinationthat occurs in the germ cells, and transplantation, cryopreservation andeventual culture of the spermatogonial stem cell provides a new andunique entry into this fundamental process.

                                      TABLE 2                                     __________________________________________________________________________    SPERMATOGENESIS FROM FROZEN TESTIS STEM CELLS TRANSPLANTED                    TO RECIPIENT MOUSE TESTES                                                                      Frozon donor                                                                         Percent testis                                                                           Number of testis                                      Time donor                                                                          cell   surface                                                                             Time to                                                                            tubules with                                     Recipient                                                                          cells frozen,                                                                       concentration                                                                        are covered.sup.                                                      analysis,                                                                          donor cells                                Experiment                                                                          mouse                                                                              days  No. × 10.sup.-6                                                                Right, Left                                                                         days Right, Left                                __________________________________________________________________________    1     887  4     28     90, 90                                                                              133  ≧12, 8                                    888  4     28     85, 90                                                                              133  0, 0                                             889  4     28     80, 90                                                                              133  0, 0                                             890  4     28     30, 25                                                                              133  0, 0                                       2     891  7     32     80, 80                                                                              124  6, ≧12                                    892  7     32     85, 75                                                                              124  5, 3                                             893  7     32     85, 45                                                                              124  3, 1                                             894  7     32     95, 70                                                                              124  ≧12, 9                              3     895  9, 13 46     85, 95                                                                              117  8, ≧12                                    896  9, 13 46     95, 95                                                                              117  ≧12, ≧12                           897  9, 13 46     55, 80                                                                              117  ≧12, ≧12                     4     771  111   18     50, 70                                                                              108  3, 8                                             789  133   12     90, 80                                                                              162  0, 5                                             791  134   15     70, 70                                                                              160  4, 0                                             798  156   11     80, 50                                                                              139  7, 5                                       __________________________________________________________________________     Recipient mice and donor cells described in FIG 1. Busulfan treatment in      experiment 1 (33 mg/kg); 2 (35-37 mg/kg), 3 (33-40 mg/kg), 4 (32 mg/kg).      Age of males supplying donor cells in experiment 4, 6-14 days; in other       experiments, 6-1 6 weeks. In experiment 3, cells frozen 9 and 13 days wer     mixed. Presence of spermatogenesis from microinjected frozen donor cells      is readily detected by staining (FIG 8).                                      .sup. Percent of surface seminiferous tubules in recipient      testes filled by donor cell suspension.sup.4.                                 .sup.+ Number of days from microinjection of cells to analysis; also time     available for spermatogenesis. Mouse spermatogenesis takes 35 days from       stem cell to mature spermatozoa.sup.6,7.                                      Number of seminiferous tubules in recipient that stain blue, indicating       the presence of spermatogenesis from donor cells. When there are more tha     1 2 tubules stained, total number cannot be accurately determined because     tubules are interwoven.                                                  

Methods

Donor cells were collected by enzymatic digestion of transgenic testes(ref. 23, 24) and were suspended in Dulbecco's modified Eagle's medium(DMEM) containing 10% fetal bovine serum (FBS), 2 mM glutamine, 6 mMlactate, 0.5 mM pyruvate, 30 mg/l penicillin, and 50 mg/l streptomycin(Designated DMEM-C); at a concentration of 16-40×10⁶ cells/ml. Freezingmedium (FBS, DMEM-C, DMSO in a ratio of 1:3:1) was added slowly by dropsto equal the original cell volume and mixed²⁵. Cells were dispensed 1.0ml per freezing vial and placed in an insulated container at -70° C. forat least 12 hr and then stored in liquid nitrogen (-196° C.). The cellswere thawed by swirling in a 37° C. water bath and DMEM-C was addedslowly by drops to 3 times the volume in the vial. Recovery of cellsfollowing freezing ranged from 47 to 61% (Mean=55), and viability of thecells ranged from 50 to 66% (Mean=60). Thus, about one-third of theoriginal cell population survived the procedure. Aggregation of cellsduring freezing and thawing was the primary cause of cell loss. Afterthawing, the cells for an experiment were pooled, centrifuged at 600×gfor 5 min at 16° C., the supernatant removed, and the pellet resuspendedin injection medium⁴. Microinjection of seminiferous tubules with donorcells and analysis of recipient mice was as previously described⁴. Usingthe techniques described in this example, rat testis cells have now beencryopreserved for up to 56 days, and they subsequently generate ratspermatogenesis when transplanted to the testis of an immunodeficientmouse (xenogeneic spermatogonial transplation procedure from reference18).

REFERENCE FOR EXAMPLE C

1. Hafez, Preservation and cryopreservation of gametes and embryos. InReproduction in Farm Animals, 6th Edition (ed. Hafez, E. S. E.) 503-525,(Lea & Febiger, Philadelphia, 1993).

2. Tada, et al., Reprod. Fert. 89, 511-516 (1990).

3. Brinster, & Zimmermann, Proc. Natl. Acad. Sci. USA 91, 11298-11302(1994).

4. Brinster & Avarbock, Proc. Natl. Acad. Sci. USA 91, 11303-11307(1994).

5. Baker, Oogenesis and ovarian development. In Reproductive Biology(eds. Balin, H. & Glasser, S.) 398-437 (Excerpta Medica, Amsterdam,1972).

6. Russell et al., Mammalian spermatogenesis. In Histological andHistopathological Evaluation of the Testis 1-40 (Cache River Press,Clearwater, 1990).

7. Ewing et al., Regulation of testicular function: A spatial andtemporal view. In International Review of Physiology (ed. Greep, R. O.)41-115 (University Park Press, Baltimore, 1980).

8. De Rooij et al., Regulation of spermatogonial proliferation. InAnnuals of The New York Academy of Science (eds. Ewing, E. E. & Robaire,B.) 140-153 (The New York Academy of Sciences, New York, 1992).

9. Gosden, Transplantation of ovaries and testes. In Fetal TissueTransplants in Medicine (ed. Edwards, R. G.) 253-280 (CambridgeUniversity Press, New York, 1992).

10. Deanesly, Endocrin. 11, 201-206 (1954).

11. Zambrowicz, et al., Development 120, 1549-1559 (1994).

12. Freshney, Instability, variation, and preservation. In Culture ofAnimal Cells, Third Edition (ed. Freshney, R. I.) 253-266 (Wiley-Liss,New York, 1994).

13. Bucci, & Meistrich, Mutation Research 176, 259-268 (1987).

14. Dym, The male reproductive system. In Histology Cell and TissueBiology, Fifth Edition (ed. Weiss, L.) 1000-1053 (Elsevier SciencePublishing Co., Inc., New York, 1993).

15. Meistrich, Nuclear morphogenesis during spermiogenesis. In MolecularBiology of the Male Reproductive System. (ed. de Kretser, D.) 67-97(Academic Press, Inc., New York, 1993).

16. Clermont et al., Cell biology of mammalian spermiogenesis. In Celland Molecular Biology of the Testis (eds. Desjardins, C. & Ewing, L. L.)332-376 (Oxford University Press, New York, 1993).

17. Setchell, Spermatogenesis and spermatozoa. In Reproduction inMammals:Germ Cells and Fertilization, Second Edition. (eds. Austin, C.R. & Short, R. V.) 63-101 (Cambridge University Press, New York, 1982).

18. Clouthier et al., Nature, In press, 1996.

19. Kimura & Yanagimachi, Development 121, 2397-2405 (1995).

20. Aitken, Nature 379, 493-495 (1996).

21. Fishel et al., The Lancet 345, 1641-1642 (1995).

22. Tesarik et al., N. Engl. J. Med. 333, 525 (1995).

23. Bellve et al., Cell Biol 74, 68-85 (1977).

24. Bellve, Purification, culture, and fractionation of spermatogeniccells. In Methods in Enzymology 225 (eds. Wassarman, P. M. &DePamphilis, M. L.) 84-113 (Academic Press, Inc., New York, 1993).

25. Robertson, Embryo-derived stem cell lines. In Teratocarcinomas andembryonic stem cells: a practical approach (ed. Robertson, E. J.) 71-112(IRL Press Limited, Oxford, 1987).

Having now fully described the invention, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionas set forth herein.

What is claimed as new and desired to be secured by Letters Patent ofthe United States is:
 1. A method of preserving spermatogoniacomprising:suspending 10⁴ to 10⁹ testis cells per ml of culture mediumcontaining a cryopreservant, pyruvate and lactate, freezing thesuspension, and storing the frozen suspension at about -180°--220° C. 2.The method of claim 1, wherein said frozen suspension is stored at -196°C.
 3. The method of claim 1, wherein said medium is Dulbecco's modifiedEagles's Medium plus 15% fetal calf serum.
 4. The method of claim 1,which comprises suspending 10⁶ cells per mL of culture medium.
 5. Aspermatogonia cell which has been cryopreserved and which can generatespermatogonia.
 6. A method of preserving spermatogoniacomprising:suspending 10⁴ to 10⁹ testis cells per ml of culture medium,wherein said culture medium is Dulbeccol's modified Eagles's Medium plus15% fetal calf serum supplemented with pyruvate and lactate andcontaining a cryopreservant, freezing the suspension, and storing thefrozen suspension at about -180°--220° C.